Phospholipase A.sub.2 s--phosphatide 2-acylhydrolase, EC 3.1.1.4 (hereinafter "PLA.sub.2 ") constitute a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphogylcerides, producing free fatty acid and lysophospholipids. See Dennis, E. A. Phospholiphases. In: The Enzymes, edited by Boyer, P. New York: Academic Press, p. 307-353 (1983). Over the past two decades, PLA.sub.2 activities have been purified and characterized from different tissues, cultured cells, and exudates from different mammals. See Rordorf, G. et al.: J. Neuroscience, 11:1829-1826 (1991); Seilhamer, J. J. et al.: J. Biochem., 106:38-42 (1989); Langlais J. et al.: Biocham. & Biophys. Res. Comm., 182:208-214 (1992); Murakami, M. et al.: J. Biochem., 111:175-181 (1992); and Jordan, L. M. et al.: J. Chromat., 597:299-308 (1992). These enzymes have been found to vary in molecular weight, optimal pH, Ca.sup.2+ dependence, substrate specificity, and solubility.
To date, two classes of unrelated PLA.sub.2 s have been reported. One is a family of low molecular mass, approximately 14 kDa PLA.sub.2 s which are characterized by a rigid three dimensional structure maintained by disulfide bridges and a catalytic requirement for Ca.sup.2+. The other is a high molecular mass, 82 kDa, intracellular PLA.sub.2 found in the cytosolic subcellular fraction in the absence of calcium, but associated with cellular membranes at calcium concentrations from 0.1 to 10 .mu.M. See Clark, J. D. et al.: Cell, 65:1043-1051 (1991) and Sharp, J. D. et al.: J. Biol. Chem., 266:14850-14853 (1991). In addition, several Ca.sup.++ -insensitive PLA.sub.2 activities are believed to exist, however, it is also believed that as yet none of the genes encoding such activities have been cloned.
In terms of structure, low molecular weight, e.g., about 14 kDa, PLA.sub.2 s rank among the best characterized enzymes. Complete primary sequences have been determined for more than 50 PLA.sub.2 s from organisms such as snakes, bees and humans. See Heinrikson, R. L.: Methods in Enzymology, 197:201-214 (1991); Davidson, F. F. et al.: J. Mol. Evolution, 31:228-238 (1990); and Dennis, E. A. Phospholiphases. In: The Enzymes, edited by Boyer, P. New York, Academic Press, p. 307-353 (1983). In all active 14 kDa PLA.sub.2 s, 18 amino acids are believed to be conserved. See Heinrikson, R. L.: Methods in Enzymology, 197:201-214 (1991) and Davidson, F. F. J. Mol. Evolution, 31:228-238 (1990). Based on selected structural determinants, the low molecular weight PLA.sub.2 s have been classified into two types. See Heinrikson, R. L. et al.: J. Biol. Chem., 252:4913-4921 (1977). Type I enzymes have a disulfide bridge connecting cysteines between amino acids 11 and 77. In addition, there is an insertion of three amino acids between residues 54 and 56, the so-called elapid loop. The only identified mammalian Type I PLA.sub.2 s, see Seilhamer, J. J. et al.: DNA, 5:519-527 (1986) and Ohara, O. et al.: J. Biochem., 99:733-739 (1986), are expressed mainly in the pancreas and function extracellularly in digestion. Type II PLA.sub.2 s, on the other hand, lack the disulfide bridge between amino acids 11 and 77, have carboxy-terminal (COOH-terminal) amino acid extensions which can vary in length, but are commonly seven amino acids in length, which typically terminate in a half-cysteine joined to Cys-50 near the catalytic site His-48. The mammalian Type II PLA.sub.2 s reported to date occur in trace amounts in several tissues such as liver and spleen and are secreted from various cells in response to appropriate stimuli. See Seilhamer, J. J. et al.: J. Biol. Chem., 264:5335-5338 (1989); Kramer, R. M. et al.: J. Biol. Chem., 264:5768-5775 (1989); Komada, M. et al.: J. Biochem., 106:545-547 (1989); Kusunoki, C. et al.: Biochimica Et Biophysica Acta, 1087:95-97 (1990); Aarsman, A. J. et al.: J. Biol. Chem., 264:10008-10014 (1989); Ono, T. et al.: J. Biol. Chem., 264:5732-5738 (1988); Horigome, K. et al.: J. Biochem., 101:53-61 (1987); Nakano, T. et al.: Febs. Letters, 261:171-174 (1990); and Schalkwijk, C. et al.: Biochem. & Biophys. Res. Comm., 174:268-272 (1991). It is believed that Type II PLA.sub.2 s are associated with the pathologies of several diseases involving infection, tissue damage, and inflammation, such as acute pancreatitis, septic shock, peritonitis and rheumatoid arthritis. See Vadas, P. et al.: Lab. Invest., 55:391-404 (1986); Pruzanski, W. et al.: Advances in Exper. Med. & Biol., 279:239-251 (1990); Uhl, W. et al.: J. Trauma, 30:1283-1290 (1990); and Malfertheiner, P. et al.: Klinische Wochenscrift, 67:183-185 (1989). Mammalian Type I and II PLA.sub.2 s share approximately 30-40% amino acid homology; however, eighteen amino acids are invariantly conserved in all known functional PLA.sub.2 s. Type I mammalian PLA.sub.2 genes contain 4 coding exons; Type II mammalian genes contain five exons, the first of which is noncoding.
In 1990, a distinct 120 bp putative PLA.sub.2 exon-like fragment (h10a), homologous to the amino-terminus encoding region of known PLA.sub.2 s, was obtained by screening a human genomic DNA library with a 45 bp human PLA.sub.2 Type II oligonucleotide probe. See Johnson, L. K. et al.: Advances in Exper. Med. & Biol., 275:17-34 (1990). Zoo blots indicated that the putative exon has been highly conserved during evolution. However, additional exons indicating the presence of a complete gene, a corresponding cDNA, or evidence of transcription in different human tissues was not found.
Neuronal ceroid lipfuscinoses (NCL), or Batten disease, are terminal, inheritable, lysosomal storage diseases of children. They are characterized by the accumulation of an autofluorescent pigment (ceroid or lipofuscin) in cells, especially neurons and epithelial pigment cells of the retina. NCL patients typically manifest high levels of the highly reactive compound, 4-hydroxynonenal. These levels are believed to be a consequence of a failure to resolve peroxidized, fatty acids in the normal way. It is believed that this failure could be the result of a phospholipase A.sub.2 defect.
The infantile form of NCL has now been linked to chromosome 1p33-35. See Jarvela, I. et al.: Genomics, 9:170-173 (1991). The non-pancreatic PLA.sub.2 (Type II) has also been mapped to chromosome 1. The Type II gene and two additional putative exon-like "fragments" (h8 and h10a), see Johnson, L. K. et al.: Advances in Exper. Med. & Biol., 275:17-34 (1990), are located at about 1p34--in the middle of the region where gene for infantile NCL is believed to reside. See Jarvala, I. et al.: Genomics, 9:170-173 (1991). h8 and h10a each contain a unique sequence which is highly homologous to, but distinct from, exon two (which contains the calcium binding domain) of PLA.sub.2 Type II.
Consequently, there is a continuing need to further identify and characterize additional PLA.sub.2 exons if such exist. Such exons could be part of unidentified genes. To the extent there are additional unidentified PLA.sub.2 exons and genes, they may encode proteins (enzymes) that function in a manner different from, similar to, or overlapping with, the known PLA.sub.2 s. Moreover, such unidentified exons and/or genes and the enzymes encoded thereby may be regulated by some of the same effectors as the known PLA.sub.2 genes and their proteins. Investigation of these unidentified exons and/or genes and their encoded enzymes may therefore result in new approaches to therapy of PLA.sub.2 -related diseases, such as Batten disease and inflammatory disease. Alternatively, these unidentified enzymes may have entirely different physiologic and pathologic functions. Thus, therapeutic approaches designed to block the activity of the known Type II PLA.sub.2 enzymes may also block or reduce the activity of these novel enzymes, thereby producing unexpected side effects. Still further, a better understanding of the regulation of expression of the known and unidentified Type II PLA.sub.2 genes in different tissues will likely expand the overall understanding of the biology and metabolic processes involving PLA.sub.2 s.